Condensed phase kinetics • The kinetic theory of gases involves ballistic motion • Kinetics in liquids and other condensed phases involves diffusion • What are the major characteristics? – Many molecules interacting at once – Short mean free paths – No “memory” of momenta Brownian motion • Extreme case of condensed phase dynamics • No memory of momentum • “Bath” provides: – Random displacements – Frictional damping • Bath action related to molecular collisions Random walks • Markov chain: no history dependence • Consider a discrete time random walk in 1D: – Equal probability of moving left or right at each time step – Position probability distribution is binomial – At a large number of steps, probability becomes Gaussian – Mean square displacement grows linearly with time • These are general features of unbiased random walks • Can be generalized to continuous time and space N! ⎛1⎞ P ( n+ , N ) = ⎜ ⎟ n+ !( N − n+ ) ! ⎝ 2 ⎠ N ⎛ 2n+2 ⎞ 2 exp ⎜ − ∼ ⎟ πN ⎝ N ⎠ ∼ m2 ∼ N 2 ⎛ m+ N) ( 1 exp ⎜ − ⎜ 2π N 2N ⎝ 1D random walk 10000 trajectories 10000 trajectories 40 0.2 30 0.15 0.1 20 0.05 x <x> 10 0 0 -0.05 -10 -0.1 -20 -30 -0.15 0 100 200 300 400 n 500 600 700 800 -0.2 10000 trajectories 800 10 0 x 10 100 200 300 5 400 n 500 600 700 800 10000 trajectories 9 700 8 600 7 <x 2> 500 6 400 5 4 300 3 200 2 100 0 1 0 100 200 300 400 n 500 600 700 800 0 -150 -100 -50 0 50 100 150 Diffusion coefficients • What is the mean squared displacement per unit time? • For unbiased random walks, it’s pretty simple • Diffusion coefficients also have microscopic interpretation… m→ x, y , z t ,N → l τ P1D ( x, t ) = ( 4π Dt ) x2 1D 2D 2D −1 ⎛ x2 + y 2 ⎞ exp ⎜ − ⎟ 4 Dt ⎠ ⎝ = 4 Dt P3D ( x, y, z , t ) = ( 4π Dt ) x2 + y2 + z 2 ⎛ x2 ⎞ exp ⎜ − ⎟ Dt 4 ⎝ ⎠ = 2 Dt P2D ( x, y, t ) = ( 4π Dt ) x2 + y2 −1 2 = 6 Dt −3 2 ⎛ x2 + y 2 + z 2 ⎞ exp ⎜ − ⎟ 4 Dt ⎝ ⎠ Langevin equation • Newton’s equation with extra terms • Random force dv ( t ) m = −ζ v ( t ) + f ( t ) dt f (t ) = 0 – Energy added by bath – Mean force is zero • Viscous drag – Energy dissipated by bath – Related to velocity decay times • The viscous drag is related to the diffusion coefficient m d v (t ) = −ζ v ( t ) dt v ( t ) = v ( 0 ) e −ζ t m Viscosity and diffusion • Solve Langevin equation for mean squared position – Multiply by x and rearrange – Use the fact that the mean squared velocity is kT (Maxwell dist.) – Solve for <xv> with particle initially at origin – Relate to <x2> and solve m d ( xv ) = −ζ xv + m v 2 dt = −ζ xv + k BT xv = k BT ζ (1 − e −ζ t m ) kT 1 d 2 x = B (1 − e −ζ t m ) ζ 2 dt x2 = 2 k BT ⎛ m −ζ t m ⎞ t 1 − − e ( ) ⎜ ⎟ ζ ⎝ ζ ⎠ Viscosity and diffusion • Two time scales for mean squared displacement – Related to “collisional time” – At short times, the motion is ballistic – At long times, the motion is Brownian • Velocity doesn’t matter • RMSD can be related to diffusion coefficient • The friction and diffusion coefficient are related! x2 = τ= x2 = lim x 2 = t τ 2 k BT ⎛ m −ζ t m ⎞ 1 − − t e ( )⎟ ζ ⎜⎝ ζ ⎠ m ζ 2 k BT ζ t τ 12 2 2 k BT ζ = 2 Dt D= −t τ k BT 2 t m = v2 lim x 2 = (t − τ (1 − e )) k BT ζ t t The Stokes-Einstein relationship • Stokes law provides an expression for the viscous drag around various simple shapes • This can be combined with the MSD derivation above • The final relationship is called the StokesEinstein relation ζ = 6πη a D= k BT ζ k BT = 6πη a Mass action kinetics • The rate of change in concentration or probability • Describes a variety of phenomena: chemical reactions, binding events, etc. • Relates changes in concentrations with time to (powers of) species concentrations • Assumes large numbers of species • Ignores fluctuations due to small copy numbers aA + bB k1 k2 P dcP ( t ) a b = k1c A ( t ) cB ( t ) − k2 cP ( t ) dt Michaelis-Menten kinetics • Three basic reactions – Substrate-enzyme association – Substrate-enzyme dissociation – Catalysis and product-enzyme dissociation • Steady-state assumption used below E+A kon koff EA ⎯⎯→ E + P kcat koff koff + kcat Kd = , KM = kon kon kcat cE ( 0 ) cS ( t ) vss = K M + cS ( t ) What is a diffusion-limited reaction? • Consider a reaction where the chemical step is instantaneous – All reactions which form ES complex go to products – The rate-limiting aspect of the reaction is binding to the enzyme • Assume low substrate concentrations E+A kcat kon koff koff , cS ( t ) kcat EA ⎯⎯→ E+P kcat kcat , KM ≈ kon kon kcat cE ( 0 ) cS ( t ) ≈ kon cE ( 0 ) cS ( t ) vss ≈ kcat + cS ( t ) kon Diffusion-limited reactions • Typical diffusional encounter rate is 109 to 1010 M-1 s-1 – There are lots of caveats, exceptions, etc.: protein flexibility, electrostatics, limited reaction surface • Smoluchowski encounter rate: – Assumes spherical symmetry – Based on solution of PDE – No interactions: proportional to sum of diffusion coefficients and separation – Interactions: related to integral of potential • • Thought to represent evolutionary pressure Examples – Superoxide dismutase – Acetylcholinesterase – Barstar-barnase w( r ) k B T ⎡ e ⎤ kD ( R ) = ⎢ ∫ dr ⎥ 2 ⎣ R 4π r D ( r ) ⎦ k D0 ( R ) = 4π DR ∞ −1 Methods for diffusional encounter simulations • Discrete methods – Langevin dynamics – Brownian dynamics – Monte Carlo • Continuum methods – Fokker-Planck – Smoluchowski equation Discrete simulations of binding events • Brownian dynamics • Use as normal dynamics methods – Integrate stochastic equations of motion – Average: configurations, thermodynamics, etc. (nothing that depends on viscosities!) • Use as encounter simulation method First-order BD integration • Calculate – Diffusion coefficient gradient – Potential of mean force gradient – Random displacement • Works for large time steps provided the gradients don’t change (much) • Position components can be x, y, z – or separate particle coordinates • Coupling between particle diffusion components: hydrodynamic interactions ri ( t + Δt ) = ri ( t ) + Δt ∑ j Ri ( Δt ) = 0 Ri ( Δt ) R j ( Δt ) = 2 Dij Δt ∂Dij ( t ) ∂rj ∂W ( t ) − Δt ∑ Dij ( t ) + Ri ( Δt ) ∂rj j BD for encounter rate calculation • Assumptions: – Low enzyme and substrate concentrations (no enzymeenzyme or substrate-substrate interactions) – Diffusion control – Implicit solvent • Basic idea: what is the probability that two molecules started at distance b will encounter one another rather than wandering off to infinity? BD for encounter rate calculation • BD trajectory: – Start two molecules at a separation b where the potential is centrosymmetric – Integrate BD equation of motion until • Molecules satisfy reaction criteria • Molecules exceed separation distance q • A maximum number of steps are taken • Perform multiple BD trajectories: – Accumulate collision frequencies – Statistics are noisy; multiple runs needed! Figure from: Northrup SH, Allison SA, McCammon JA. J Chem Phys 80 (4) 1517-24, 1984. BD for encounter rate calculation BD for diffusion-limited reactions • Collision frequencies can be transformed into rates • Think: flux through reactive site! • If all collisions result in reaction (diffusion-limited), rate is related to: – Rate of diffusion to separation b (can use Smoluchowski formula) – Collision frequency – Probability that trajectories leaving q returns to b kD ( b ) β k= 1 − (1 − β ) Ω w( r ) k B T ⎡ e ⎤ kD ( b ) = ⎢ ∫ dr ⎥ 2 ⎣ b 4π D ( r ) r ⎦ ∞ ∞ Ω= e w( r ) k B T ∫ 4π D ( r ) r q ∞ e dr 2 dr w( r ) k B T ∫ 4π D ( r ) r b 2 −1 BD for diffusion-influenced reactions • If only some collisions result in reaction (probability α), rate is related to: – All of above – Reaction probability α – Probability Δ that unsuccessful encounter results in later collision ⎡ ⎤ β α kD ( b ) ⎢ ⎥ 1 − (1 − β ) Ω ⎦ ⎣ k= ⎧⎪ ⎡ ⎤ ⎫⎪ β 1 − (1 − α ) ⎨Δ + (1 − Δ ) ⎢ ⎥⎬ ⎪⎩ ⎣1 − (1 − β ) Ω ⎦ ⎪⎭ Interactions in BD calculations • Forces – Long-range influences only – Electrostatics: approximate charge-field calculations Filig ≈ qilig E prot • Poisson-Boltzmann calculation for protein, charge model for ligand • No desolvation • Little “internal dielectric” screening (some effective charge methods) • Diffusion coefficients – Should include rotation, translation, and configuration changes – No hydrodynamic interactions • Probably OK for small ligands • Stokes-Einstein isotropic diffusion coefficients • Coefficients do not depend on distance or configuration – Hydrodynamic interactions • Include water-mediated effects • Oseen and other (approximate) analytic forms • Configuration- and distancedependent αβ Dij 1 − δ ij k BT ⎛ δ ij ≈ ⎜ I+ cπη ⎜⎝ ai 2 Rij ⎧ai + a j Rij = ⎨ ⎩ rij ⎛ rij rij ⎜⎜ I + 2 rij ⎝ rij < ai + a j rij ≥ ai + a j ⎞⎞ ⎟ ⎟⎟ ⎟ ⎠⎠ Application to acetylcholinesterase • • • Hydrolytic enzyme in neuromuscular junction Subject of extensive computational (BD) and experimental study Properties: – Diffusion-limited catalysis – Long, narrow active site gorge – Significant electrostatic influences AChE/TMA binding • Binding of neurotransmitterlike molecule to acetylcholinesterase • Diffusion-controlled binding • Significant dependence on [NaCl] • Sensitivity to charged residues Figures from: Tara S, et al. Biopolymers 46 (7) 465-74, 1998. DHFR-TS substrate channeling • • Bifunctional enzyme: thymidylate synthase produces dihydrofolate used by dihydrofolate reductase Electrostatic steering between active sites enhances efficiency – Reduces diffusional broadening – Does not “direct” between sites Figures from: Elcock AH, et al. J Mol Biol 262, 370-4, 1996. Protein-protein encounter • • • Same basic procedure as before Reaction criteria are harder to evaluate – often a variable in the simulation Effective charge method – – • Use full electrostatic grid for one molecule Use a smaller number of charges: termini and charged residues Usually neglect: – – – – – Desolvation terms Hydrodynamic interactions (with exceptions) Ion relaxation Flexibility Substrate-substrate interactions Figures from: Gabdoulline RR, Wade RC. J Phys Chem 100 3868-78, 1996 and ibid. Methods 14 329-41, 1998. Actin polymerization • BD simulations of actin polymerization • Reproduced experimental observation of faster polymerization at “barbed” filament end • Implicated electrostatics in faster binding to barbed end • Also observed effect of ADFcofilin on polymerization Figures from: Sept D, Elcock AH, McCammon JA. J Mol Biol 294 (5) 1181-9, 1999. Lots of protein-protein association rates • Systems studied: – – – – • • • • Barstar-barnase AChE-Fas2 Cyt C peroxidase-Cyt C HyHEL antibodies and lysozyme Agreement with experiment is good Antibody/lysozyme and AChE-Fas2 rates overestimated: not diffusion-limited? Electrostatics aren’t always helpful! Figures from: Gabdoulline RR, Wade RC. J Mol Biol 306 1139-55, 2001. Continuum diffusion simulation methods • Discrete methods – Solve stochastic ODEs – Provide atomic problem resolution – Facilitate integration of stochastic phenomena – Software: MCell, UHBD, etc. • Continuum methods – Solve deterministic PDEs – Bridge larger length scales – Facilitate integration of continuum mechanics phenomena – Software: SMOL Continuum diffusion motivation • Demonstrate: – Accurate description of enzyme binding kinetics (steadystate and time-dependent) – Simulation of synapse electrophysiology – Extreme adaptivity of methods to bridge length scales • Long-term goals: – Integrate continuum and discrete methods – More complete description of cellular-scale processes Smoluchowski equation Concentration change over time Diffusion term Drift term ∂ρ ( x) = ∇ ⋅ J ( x) = ∇ ⋅ D( x)[∇ρ ( x) + βρ ( x)∇W ( x)] ∂t Flux Diffusion External coefficient ρ (x) = ρ “Bulk” boundary condition ρ ( x) = 0 Reactive boundary condition n( x ) ⋅ J ( x ) = 0 Reflective boundary condition k (t ) = ∫ J ( s ) ⋅ n( s )ds The observable: the time-dependent rate constant potential Advances and outlook • Receptor flexibility • Detailed binding mechanisms • Imperfect reactivity; calculate “re-entrant” trajectories HEL-antibody flexibility in lysozyme binding from Gabdoulline RR, Wade RC. J Mol Biol 306 113955, 2001. Camphor release pathway from cytochrome P450 from Guallar lab. Advances and outlook • Cellular simulations • Proteomics-scale interactions • Crowded environments Virtual Cell schematic from Slepchenko BM, Schaff JC, Carson JH, Loew LM. Annu Rev Biophys Biomol Struct 31 423-41, 2002. Crowding and GroEL simulation from Elcock AH. PNAS 100 (5) 2340-4, 2003.